IN VITRO BIOSYNTHESIS OF 18-HYDROXY-11-DEOXYCORTI- COSTERONE FROM DEOXYCORTICOSTERONE BY HUMAN ADRENAL GLANDS REMOVED FROM PATIENTS WITH HYPERCORTICISM
Y. TOUITOU, A. BOGDAN and J-P. LUTON
Biochemical Department, Faculty of Medicine Pitié-Salpêtrière, Paris and Department of Endocrinology, Hôpital Cochin, Paris.
(Received 10 October 1978; revised 27 November 1978; accepted 3 December 1978)
SUMMARY
The biosynthesis of 18-hydroxy-11-deoxycorticosterone (18-OH-DOC) was studied by in vitro incubations of human adrenals resected from patients with Cushing’s syndrome, using deoxycorticosterone as precursor. 18-OH-DOC synthesis was similar in adrenals from normal subjects and in those derived from patients with different types of Cushing’s syndrome (hyperplasia, adenoma, carcinoma). The in vitro addi- tion of o,p’-DDD (5 X 10-3 M) to an adrenocortical carcinoma incubation did not alter 18-OH-DOC biosynthesis. By contrast, treatment of the patient with o,p’-DDD prior to adrenalectomy resulted in a low production of 18-OH-DOC in vitro but only when cortisol synthesis was drastically decreased.
Peron (1961) and Birmingham & Ward (1961) found significant amounts of 18-hydroxy- deoxycorticosterone (18-OH-DOC) in incubated rat adrenal slices. Since then, evidence has been obtained for the synthesis of this steroid in the adrenal of the camel (Race & Wu, 1964), frog (Kraulis & Birmingham, 1964), trout (Arai & Tamaoki, 1967), rabbit (Fazekas & Kokai, 1967) and human (Carballeira & Venning, 1967). 18-hydroxydeoxycorticosterone secretion rates in man have been well documented (for a review see Melby et al., 1972). By contrast, few data are available on the in vitro synthesis of this steroid from deoxycorticosterone (DOC) as a precursor in human adrenals resected for Cushing’s syndrome. This report presents the results obtained with a number of such adrenals removed at surgery from patients, some of whom had been previously treated with o,p’-DDD.
MATERIAL AND METHODS
Chemicals. [1,2-3H] deoxycorticosterone (SA = 46.8 Ci/m.mol), [1,2-3H] 17a hydroxy- 11-deoxycorticosterone (SA = 44.1 Ci/m.mol), [4-14C] cortisol (SA = 55.2 mCi/m.mol)
Correspondence: Dr Y. Touitou, Biochemical Department, Faculty of Medicine Pitié-Salpêtrière, 91 Boulevard de l’Hôpital, 75634 Paris Cedex 13.
0300-0664/79/0010-0039$02.00 @ 1979 Blackwell Scientific Publications
were purchased from the New England Nuclear Corporation. The radiochemical purity of these steroids was checked by paper chromatography shortly before use. [4.14C] 18-hydroxy- 11-deoxycorticosterone was obtained from [4.14C] deoxycorticosterone by rat adrenal homogenate incubation. The radiochemical purity was checked according to a method des- cribed elsewhere (Touitou et al., 1975). Nicotinamide adeninedinucleotide phosphate (NADP+) and potassium hydroxide were purchased from Merck, malic acid from Sigma, and sterile Earle’s medium from Institut Pasteur, Paris. The solvents were analytical grade from Merck.
o,p’.DDD: 2(0-chlorophenyl)-2 (p-chlorophenyl)-1,1-dichloroethane, was generously supplied by Roussel Pharmaceutical Corporation, Paris.
Animal and human adrenals
Rat adrenals: They were removed immediately after death and brought to the laboratory in an ice-filled container. The homogenate was prepared with the pooled glands of twenty animals.
Human adrenals: Adrenals were removed from female patients suffering from Cushing’s syndrome. In all, six adrenals were studied in vitro. Two adrenals (one hyperplasia and one adrenocortical carcinoma) were resected from untreated patients, whereas two adrenals (one adrenocortical adenoma and one hyperplasia) were removed from o,p’-DDD-treated patients. These patients (cases 3 and 4) received o,p’-DDD orally for 3 months (total dose: 1180 g) and 12 months (total dose: 2340 g) respectively. Finally, one of the patients (case 5) was submitted to bilateral adrenalectomy with a 4-month interval between removal of the two glands. She received o,p’-DDD for 1 month, at a total dose of 324 g just prior to the first adrenalectomy. She was not treated prior to the second adrenalectomy. In all cases, treat- ment was always stopped on the day before surgery.
Adrenals glands were brought to the laboratory in ice-filled containers and frozen at - 20°℃ until processed.
Table 1 displays the main morphological features of the adrenals as well the scheme of treatment.
Experimental procedure
Adrenal glands: The adrenal glands of both humans and animals were cleared of fat and connective tissue. The adrenal tissue was weighed, trimmed and homogenized with a Teflon glass homogenizer in Earle’s medium buffer. The calcium concentration of the medium was adjusted to 4 mM by adding CaCl2. Human adrenal incubations were performed in duplicate with a tissue weight ranging between 200 mg and 1 g. Each animal adrenal incubation (n = 4) contained 115 mg, wet weight, of adrenal homogenates prepared from the pooled glands of 20 rats. Incubation flasks contained exactly measured amounts of radioactive precursors: tritiated deoxycorticosterone or 11-deoxycortisol and an NADPH generating system made up with NADP+ (1 mM) and malic acid (5 mM) neutralized with a 0.1 N potassium hydroxide solution (Touitou et al., 1970). The final volume of each incubation flask was 10 ml.
o,p’-DDD addition in the medium: 50 umol of o,p’-DDD were added directly as a powder to incubation flasks (final concentration = 5 × 10-3 M).
Incubation: Aerobic incubations were performed in a Dubnoff metabolic shaking incu- bator at 37℃ for 2 h. At the end of the incubation, 15.0 ml acetone were added to stop enzymatic reactions by precipitating the proteins. In order to account for procedural losses during subsequent extraction and identification of synthesized steroids, a trace amount of
| Patients | Age | Adrenal pathology | Histological details | Localization | Adrenal weight (g) | Medulla |
|---|---|---|---|---|---|---|
| Untreated Patients | ||||||
| 1-Gr. | 41 | Hyperplasia | 1 extracapsular nodule | Left | 9.60 | N |
| 2-Ma. | 26 | Carcinoma | Haemorrhagic and necrotic foci | Left | 700 | - |
| Treated Patients | ||||||
| 3-Or. | 38 | Adenoma | Spongiocyte cells | Left | 12 | ZZ |
| 4-Mi. | 16 | Hyperplasia | Moderate hyperplasia | Right | 7.50 | |
| Patient alternately treated and not | ||||||
| 5-Ja. treated with o,p'-DDD | 44 | Hyperplasia | 1 extracapsular nodule | Left | 8 | |
| 5 (2nd operation)-Ja. untreated with o,p'-DDD | 44 | Hyperplasia | Micronodule | Right | 8 | ZZ |
[4-14C] 18-hydroxy-11-deoxycorticosterone and [4.14C] cortisol was added to the incuba- tion flasks. Extraction of steroids was performed as described previously (Touitou et al., 1975).
Steroid isolation and characterization: The following solvent systems were used for descending paper chromatography (PC) of labelled steroids and conversion products: PC 1 : dichloroethane/ethylene-glycol; PC 2: toluene/propylene-glycol; PC 3: benzene/formamide; PC 4: benzene-heptane/methanol-water (67-33/80-20); PC 5: benzene/methanol-water (10/5-5).
Radioactive material was detected on paper chromatograms with a radiochromatogram scanner (Packard, model 7200). Steroid separation was achieved by descending paper chromatography. Individual metabolites isolated were further run in suitable chromatographic systems in which their isopolarity with carbon-14 internal standards was established. A con- version product was considered pure when constancy of 3H/14℃ ratios was established in successive chromatographic systems. The dried extracts with 11-deoxycortisol as a precursor were first chromatographed in a PC 1 system for 4 h, which allowed the separation of cortisol and unreacted 11-deoxycortisol.
18-hydroxy-11-deoxycorticosterone was first separated from the other steroids in PC 2 system for 7 h. The steroid was then purified in PC 3 system for 24 h, then characterized by periodic acid oxidation into the lactone of 18-hydroxy-11-deoxycorticosterone, the polarity of which was checked in PC 3 and PC 4 systems.
Cortisol was purified in PC 1 system for 17 h, then characterized by chromic acid oxida- tion into 11-ketoandrostenedione, the polarity of which was checked in PC 4 and PC 5 systems.
RESULTS
Incubations with rat adrenal homogenates in the presence of deoxycorticosterone as pre- cursor yield 15.9% as a mean (range: 15.7% to 16.2%; n = 4) of 18-hydroxydeoxycorti- costerone expressed as a percentage of the incubated precursor.
The incubation of adrenals, one carcinoma and one micro-adenomatous hyperplasia, from subjects who had not undergone any treatment with an inhibitor of adrenal steroidogenesis, with desoxycorticosterone as a precursor showed a 1.86% conversion rate to 18-hydroxy- deoxycorticosterone (range 1.45% to 2.28%). The in vitro addition of 5 X 10-3 M o,p’-DDD to the adrenocortical carcinoma (case 2) incubation did not alter the 18-hydroxydeoxycorti- costerone synthesis.
Conversion rates of 11-deoxycortisol to cortisol were always high in adrenals removed from patients without previous treatment (range 34.1% to 64.4%) and markedly lower with the adrenals resected from treated patients (1.18% to 3.36%). In one instance, the data could be obtained from the incubation of both adrenals from the same patient: the first adrenal was removed after treatment with o,p’-DDD (case 5) while the second one (case 5, 2nd opera- tion) was removed after a 4 month period without any treatment; this adrenal can be considered as a control when compared to the first one. Despite the notable decrease in 11- deoxycortisol to cortisol conversion rate, there was no significant change in deoxycorti- costerone to 18-hydroxydeoxycorticosterone conversion rate. By contrast, in the two other patients (cases 3 and 4), 18-hydroxydeoxycorticosterone synthesis was presumably lowered by o,p’-DDD treatment (0.23 to 0.26%).
Table 2 displays the data obtained with human Cushing’s type adrenals.
| Adrenals | S to F conversion | DOC to 18-OH DOC conversion | Treatment with Daily dose Duration | O'P'-DDD Total dose (g) | |||
|---|---|---|---|---|---|---|---|
| pmol F | % F | pmol 18-OH DOC | % 18-OH DOC | (g) | (month) | ||
| Untreated Patients | |||||||
| 1-Gr. | 23.8 | 34.1 | 2.06 | 1.45 | |||
| 2-Ma. | 44.9 | 64.4 | 3.24 | 2.28 | |||
| id. + o,p'-DDD | 43.1 | 61.2 | 3.40 | 2.35 | |||
| (5 × 10-3 M) | |||||||
| Treated Patients | |||||||
| 3-Or | 2.34 | 3.36 | 0.37 | 0.26 | 12 | 3 | 1180 |
| 4-Mi | 0.82 | 1.18 | 0.33 | 0.23 | 6 | 12 | 2340 |
| Patient alternately treated and not | |||||||
| 5-Ja treated with o,p'DDA | 5.3 | 22.4 | 3.76 | 2.65 | 12 | 1 | 324 |
| 5 second operation-Ja untreated with o,p'DDD | 44.6 | 64.0 | 2.99 | 2.11 | |||
DISCUSSION
18-hydroxy-11-deoxycorticosterone is known to be one of the principal corticosteroids of rat adrenal (Birmingham & Ward, 1961; Peron, 1961). The data that were obtained, in the experimental conditions, by incubating rat adrenal homogenates were in close agreement with those obtained by Tsang & Stachenko (1969) and Melby et al. (1972), with the same animal adrenals and the same precursor.
The goal of our present work was to study the biosynthesis of 18-hydroxydeoxycorti- costerone in adrenals removed from patients suffering from Cushing’s syndrome, some of whom were treated with an inhibitor of adrenal steroid synthesis, o,p’-DDD.
The following conclusions can be drawn from the results obtained in this report:
(1) No marked difference in 18-hydroxydeoxycorticosterone synthesis in vitro can be seen between the different Cushing’s type adrenals. A striking point is the lack of a significant difference between the deoxycorticosterone to 18-hydroxydeoxycorticosterone conversion rates of adrenal tissues as different as an adrenal cortical carcinoma (2.3% as a mean) and adrenal hyperplasia (1.8% as a mean).
The biosynthesis of 18-hydroxydeoxycorticosterone in vitro has been documented from deoxycorticosterone as a precursor in human adrenals removed from patients with breast carcinoma by De Nicola et al. (1970) (conversion rate: 0.14%-1.42%), or from a patient with a prostatic carcinoma by De Nicola & Birmingham (1968) (conversion rate: 1.24%), or from a normal adrenal tissue adjacent to an adrenocortical carcinoma by Ulick (1976), with progesterone as a precursor (conversion rate: 1.24%).
It has to be noted that there was no marked difference in 18-hydroxydeoxycorticosterone biosynthesis in vitro between our data on Cushing’s type adrenals removed from untreated patients and the aforementioned human adrenal tissues despite the variety of pathology.
(2) No significant changes in the in vitro synthesis of 18-hydroxydeoxycorticosterone was found when o,p’-DDD (5 X 10-3 M) was added to the incubation media. This data completes our findings on the lack of effect of o,p’-DDD in vitro on cortisol, 18-hydroxycorticosterone and aldosterone synthesis by sheep adrenals (Touitou and Bogdan, 1978) and on cortisol synthesis by normal human adrenals in vitro (Touitou et al., 1978).
(3) With all the incubated Cushing’s adrenals (whether treated or untreated) yields of cortisol from 11-deoxycortisol have been documented as an index of the enzymatic activity of the glands, as well as an index of the treatment efficiency. While in untreated patients’ adrenals the in vitro cortisol synthesis was marked (up to 64%), it was markedly lower (1.18%-3.36%) in the adrenals removed from o,p’-DDD-treated patients (cases 3 and 4), thus suggesting an effect of o,p’-DDD on 110-hydroxylase activity in vitro after in vivo administration, as reported in a recent paper by Touitou et al. (1978). It has to be noted that in these two instances (cases 3 and 4) deoxycorticosterone to 18-hydroxydeoxycorticosterone conversion rates were presumably lowered when compared with the untreated patients’ adrenals. More- over, there was no difference in the synthesis of 18-hydroxydeoxycorticosterone, whatever the histological features of the studied adrenals (adenoma or hyperplasia), after in vivo treat- ment with o,p’-DDD.
(4) In the two adrenals (cases 5 and 5, 2nd operation) obtained from the same patient, one after treatment with o,p’-DDD and the other without any preoperative treatment, no signi- ficant change in 18-hydroxydeoxycorticosterone synthesis from deoxycorticosterone could be seen despite a noticeable decrease in the cortisol synthesis in the treated adrenal when compared with the untreated one.
However, it has to be emphasized that cortisol synthesis in this treated adrenal (case 5), although much lower than that of its control (case 5, 2nd study), can be regarded as still high (conversion rate: 22.4%) when compared with the other treated adrenals (cases 3 and 4; con- version rates: 1.18 and 3.36%). This suggests the possibility of a better efficiency of the pre- surgical treatment with o,p’-DDD in these two latter adrenals.
In conclusion, the present study shows that the biosynthesis of 18-OH-DOC in Cushing’s type adrenals was very similar to that of other types of human adrenals, normal or abnormal. Cortisol and 18-hydroxy-1 1-deoxycorticosterone are both known to be ACTH-dependent in vivo (Melby et al., 1972). Our study thus raises the problem of the in vitro dissociation between the increased biosynthesis of cortisol and the unexpectedly normal biosynthesis of 18-OH-DOC in adrenals removed from untreated patients with Cushing’s syndrome.
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